Evaluation of allelopathic effects of Parthenium hysterophorus L. methanolic extracts on some selected plants and weeds

Herbicides made from natural molecules are cost-effective and environmentally friendly alternatives to synthetic chemical herbicides for controlling weeds in the crop field. In this context, an investigation was carried out to ascertain the allelopathic potential of Parthenium hysterophorus L. as well as to identify its phenolic components which are responsible for the allelopathic effect. During the observation, the rate of germination and seedlings’ growth of Vigna subterranea (L.) Verdc, Raphanus sativus (L.) Domin, Cucurbita maxima Duchesne., Cucumis sativus L., Solanum lycopersicum L., Capsicum frutescens L., Zea mays L., Abelmoschus esculentus (L.) Moench, Daucus carota L., Digitaria sanguinalis (L.) Scop and Eleusine indica (L.) Gaertn were investigated by using methanol extracts, isolated from leaf, stem and flower of P. hysterophorus. Six concentrations (i.e., 25, 50, 75, 100, and 150 g L-1) of methanol extracts were isolated from P. hysterophorus leaf, stem and flower were compared to the control (distilled water). It was also observed that the concentration of methanol extracts (isolated from P. hysterophorus leaf, stem, and flower) while increased, the rate of seed germination and seedling growth of both selected crops and weeds decreased drastically, indicating that these methanol extracts have allelopathic potential. The allelopathic potential of P. hysterophorus leaf extraction (811) was found higher than the extraction of the stem (1554) and flower (1109), which is confirmed by EC50 values. The principal component analysis (PCA) was also used to re-validate the allelopathic potentiality of these methanol extracts and confirmed that Raphanus sativus, Solanum lycopersicum, Capsicum frutescens, Abelmoschus esculentus, Daucus carota, Digitaria sanguinalis, and Eleusine indica were highly susceptible to allelochemicals of P. hysterophorus. Besides these, the LC-MS analysis also revealed that the P. hysterophorus leaf extract contained 7 phenolic compounds which were responsible for the inhibition of tested crops and weeds through allelopathic effect. The results of the current study revealed that the leaf of P. hysterophorus is a major source of allelopathic potential on crops and weeds and which could be used as a valuable natural herbicide in the future for the sustainability of crop production through controlling weeds.


Introduction
Parthenium hysterophorus (L.) is a noxious herb that has now invaded 46 countries and extended its spread from a few islands to around the world, there have been eleven minor and eight major introductions [1]. Its high invasiveness is associated with several factors, including a higher number of seeds production, highly competitive and rapidly expanding, biological plasticity of the life cycle, allelopathic ability and high survival ability against biotic and abiotic stresses [2][3][4][5].
P. hysterophorus is a species of flowering plant in the family Asteraceae. Originally, it was found in the Gulf of Mexico, the USA, the West Indies, and Central America [6]. It is a fastmaturing annual weed with a deep taproot and an erect stem that becomes woody with age. Due to its allelopathic influence, this weed is thought to cause allergic respiratory problems, mutagenicity in humans and livestock, and severe reductions in crop production [7]. Its an annual herbaceous plant that reproduces mostly through seeds. After sprouting, the young plant produces a basal rosette of finely lobed, bright green leaves that are 8-20 cm long and 4-8 cm wide. The rosette stage can continue to grow under unfavourable circumstances and reach a maximum length of 2.5 m [8]. The flower heads are terminal and somewhat hairy; they consist of several small white capitula-shaped florets. Usually, each head has five productive ray florets, although occasionally six or eight. Thousands of branches, which develop in separate clusters, produce compressed black seeds about 2 mm in size [9].
Allelopathy is described as chemicals' positive or negative impacts on substances formed primarily by plant, microbe, and fungal secondary metabolism on the growth and establishment of neighbouring plants or microorganisms, as well as the dynamical processes of agricultural and natural ecosystems [10]. It's a complicated phenomenon that's influenced by a variety of internal and external circumstances. Due to its intricacy, the explanation is a difficult endeavour that necessitates knowledge from a variety of professions [11]. Allelochemicals are plants that release secondary metabolites into the environment. They are anti-inflammatory substances that belong to a variety of chemical classes, primarily phenolic compounds and terpenoids [12]. A comprehensive summary of allelochemicals' that affects plant growth and development was also observed by Bhadoria [13]. All plant organs (stems, leaves, rhizomes, roots, flowers, pollen, fruits, and seeds) contain allelochemicals, which are released through volatilization, leaf leaching, plant material breakdown, and root exudation. In some way, membrane stability, cell division, elongation, shape and permeability, enzyme activity, and respiration of plants are all influenced. Photosynthesis, protein synthesis, nucleic acid metabolism, and other direct and indirect ways of action cause seed sprouting suppression and limited seedling development [12]. In addition, the microbial breakdown of soil allelochemicals has an impact on the effective dose of allelochemicals that can inhibit plants [14,15].
Herbicides have been the least expensive and principal method of weed control in developing countries for about 50 years [16]. Herbicides, on the other hand, pose significant risks to agriculture, human health, and the environment. However, increasing crop production without using chemical herbicides is an urgent challenge in crop production. Manual weed management is the most effective and long-term solution for weed management. So, accurate weed control is necessary for food security throughout the world. Therefore, researchers are motivated to seek alternatives because of the labour movement from agriculture to others, and weed biotypes resistant to traditional synthetic pesticides [17]. This strategy will aid in reducing reliance on chemical herbicides, reducing the likelihood of weed resistance to herbicides, reducing health risks and environmental damage, and strengthening the national economy. In the meantime, there are a variety of possible allelochemicals in aerial sections (e.g. leaves) of Parthenium weed have been confirmed by several earlier studies; among them p-anisic acid (C 8 [18,19] sprouting and development of a plant species in abundance, natural plants are included and different crops and pasture species can be inhibited by these chemicals [20]. Wheat, maize and horse gram [5], lentil [21,22] and other field crops. Hassan et al. [23] showed an inhibitory impact when exposed to parthenium extract. Dhawan and Gupta [19] reported that the extraction of diverse active phytochemicals with flavonoid concentrations works best using methanol as an extraction solvent. However, there is insufficient evidence of the allelopathic potential of Parthenium methanolic extracts on the sprouting and seedlings development of several crops, particularly bambara groundnut weeds. The bambara groundnut is a new crop for Malaysia, but there is information lacking on the suppression of allelopathy on Bambara groundnut weeds by different parts of P. hysterophorus. The current study aimed to find out the allelopathic capacity of Parthenium in a laboratory experiment to evaluate the allelopathic suppression of weeds by P. hysterophorus in Bambara groundnut weeds. The research was directed with the following objectives (1) to evaluate the Allelopathic potential of methanol extracts made from the aerial portions of P. hysterophorus on target species to develop bioherbicides based on natural products (2) LC-MS was used to identify its phenolic derivatives.

Experimental location
Growth chamber research was carried out at Weed Science Lab in the Crop Science Department, Faculty of Agriculture, Universiti Putra Malaysia (3˚02' N, 101˚42' E, elevation 31 m), Malaysia. The temperature in the growth chamber was maintained at 25˚C throughout the experimental period. laboratory blender, both bulked plant components were ground into fine dust and sieved through a 40-mesh sieve.
The inhibitory action of P. hysterophorus was investigated on nine plant species. Bambara groundnut (Vigna subterranea L. Verdc), radish (Raphanus sativus L. Domin), sweet gourd (Cucurbita maxima Duchesne), tomato (Solanum lycopersicum L.), cucumber (Cucumis sativus L.), pepper (Capsicum frutescens L.), maize (Zea mays L.), carrot (Daucus carota L.) and okra (Abelmoschus esculentus L. Moench) and two weed species goosegrass (Eleusine indica L. Gaertn)] and [crab grass (Digitaria sanguinalis L. Scop). Crop seeds were attained from Sin Seng Huat Seeds Sdn Bhd Company in Malaysia, while seeds of grasses were personally picked from the Universiti Putra Malaysia's agricultural field. The seeds were cleaned, air-dried, and stored in airtight containers maintain at -18˚C. The vegetable crops are chosen for the determination of ecological effects of allelopathic substances as they represented commonly used species in the field that are recommended by US EPA [25]. They belong to different plant families and can provide great genetic diversity. The seeds germinated 86-95% of the time, according to a random test.

Extract preparation
The extracts were made according to the procedure published by [23,26]. Accurately 100 g powder from leaves, stems, and flowers of parthenium was placed in a conical flask and allowed to soak in 1L of 80% (v/v) methanol separately. After that, the conical flask was wrapped in paraffin and shaken for 48 hours at 24-26˚C room temperatures in an Orbital shaker at 150 rpm agitation speed. To remove debris, cheesecloth in four layers was used to filter the mixtures and centrifuged for one hour at 3000 rpm in a centrifuge (5804/5804 R, Eppendorf, Germany). A single layer of Whatman No. 42 filter paper was used to filter the supernatant. A 0.2-mm Nalgene filter was used to filter the solutions once more to avoid microbial development (Lincoln Park, NJ-based Becton Dickinson percent Labware). Using a rotary evaporator (R 124, Buchi Rotary Evaporator, Germany), the solvents were evaporated from the extract to dryness (a thick mass of coagulated liquid) under vacuum at 40˚C and the sample was then collected. From a 100 g sample of P. hysterophorus powder, the average extracted sample was 17.56 g, which was estimated as per the following formula [1].
For the bioassay, each stock extract from P. hysterophorus leaves, stems and flowers were diluted in sterile distilled water to provide extract concentrations of 25, 50, 75, 100, and 150 g L -1 , while purified water was served as control. All extracts were stored at 4˚C in the dark until use.
For LC-MS analysis, 100% HPLC GRADE methanol (20 mL) was diluted with the crude sample (20 mg) and filtered through 15-mm, 0.2-μm syringe filters (Phenex, Non-sterile, Luer/ Slip, LT Resources Malaysia). Six phenolic derivatives were isolated from methanol extract of Parthenium hysterophorus in different parts through LC-MS analysis which has been discussed in detail in Table 1.

Germination and growth bioassays
Healthy, uniform seeds were gathered and treated with 0.2% potassium nitrate for 24 hours (KNO 3 ) before being rinsed with distilled water. Twenty Bambara groundnut and sweet gourd seeds and thirty seeds of radish, cucumber, tomato, pepper, maize, okra, carrot, crabgrass, and goosegrass were set up in a sterilized Petri dish with Whatman No. 1 filter paper (90×15 mm). 10 mL of extract of each concentration (25,50,75, 100 and 150 g L -1 ) was delivered in Petri dishes, distilled water serving as a control. In a growth chamber, all Petri dishes were inserted. and incubated at 30˚C/20˚C (day/night) temperature under fluorescent light (8500 lux) on photoperiod 12 h day/12 h night maintaining 30-50% relative humidity. To facilitate gas exchange, the petri dish lids were not sealed.

Identification of phenolic derivatives in P. hysterophorus leaves, stems, and flowers extracted in methanol
The LC-MS was used to identify the chemical contents of the extracts. The phytochemical compounds of the methanol extracts were performed using LC-MS followed by [31]. LC-MS analysis was performed using Agilent spectrometry equipped with a binary pump. The LC-MS was interfaced with Agilent 1290 Infinity LC system coupled to Agilent 6520 accurate-mass Q-TOF mass spectrometer with a dual ESI source. Full-scan mode from m/z 50 to 500 was performed with a source temperature of 125˚C.
The column of Agilent zorbax eclipse XDB-C18, narrow-bore 2.1x150 mm, 3.5 microns (P/ N: 930990-902) was used with the temperature 30˚C for the analysis. A-0.1% formic acid in water and B -0.1% formic acid in methanol were used as solvents. Isocratic elution was used to supply solvents at a total flow rate of 0.1 mL minutes -1 . MS spectra were collected in both positive and negative ion modes. The drying gas was 300˚C, with a 10L min-1 gas flow rate and a 45-psi nebulizing pressure. Before analysis, 1 ml of concentration. sample extracts were diluted with methanol and filtered through a 0.22 m nylon filter. The extracts were injected into the analytical column in 1 μl volume for analysis. The mass fragmentations were discovered using an Agilent mass hunter qualitative analysis B.07.00 (Metabolom-ics-2019.m) tool and a spectrum database for organic chemicals.

Data collection
The germination percentage, radicle, and hypocotyl length were measured with a ruler at seven days after seeding. The radicle and hypocotyl length was assessed by software Image J [32] while the inhibition (%) of P. hysterophorus extracts on a radicle, and hypocotyl length was computed following the formula used by Kordali [33]: Here, "I" is the percentage of inhibition, "C" control's mean growth and development and "A" is the aqueous extracts' mean growth and development.

Statistical analysis
On pooled (two seasons) data, a one-way analysis of variance (ANOVA) was used to regulate any significant variances among concentrations and control. To calculate the difference between the concentration means, the Tukey test (SAS 9.4) with a 0.05 probability level was utilised. EC r50 , EC g50 , and EC h50 were used to compute real dosages accomplished of suppressing 50% of germination, radicle development, and hypocotyl growth. Based on the suppression of germination (percentage), radicle, and hypocotyl development, Probit analysis was used to compute the EC g50 , EC r50 , and EC h50 values. From each tested plant, a rank was determined by using the following equation to calculate an index (R e ) for each of the most active extracts and plants that are the most susceptible: Where Re is the plant's rank n, EC r50n , EC h50n and EC g50n are the amounts of plant extract n that inhibit 50% germination, radicle, and hypocotyl length, respectively. The lowest Re value had the maximum active tissue extracts and the utmost sensitive plants, while the highest Re value had the least allelopathic effect of the extract.
The most common application of NTSYSpc 2.02e (Numerical Taxonomy and Multivariate Analysis System) is to do various types of agglomerative cluster analysis of some type of similarity or dissimilarity matrix and the quantity of extract sensitivity among the plants under investigation [34,35]. The principal component analysis (PCA) was used to re-validate Johnson's cluster analysis [36].

Inhibitory influence of P. hysterophorus on crop species
Different concentrations of methanolic extracts in the control, Parthenium leaf, stem, and flower concentrations and different crops had a significant influence on the germination of seed, radicle, and hypocotyl length of the examined plants, as well as a rise in extract concentration. Parthenium extracts had a bit stimulatory impact on seed germination at 25 g L -1 , but an inhibitory effect was observed at higher dosages (Fig 1).
Methanolic extract of the leaf at 25 g L -1 significantly decreased the sprouting of all plants except sweet gourd, cucumber, and maize (p�0.05), while, seed germination failure was seen in tomato, carrot, and goosegrass if the concentration level further increased. The maximum concentration resulted in 100% germination failure in all crops except cucumber (76%) and maize (65%) (Figs 1 & 2). When the P. hysterophorus stem and flower extract were applied at lower doses (25, 50, and 75 g L -1 ), there was no significant reduction in germination (%). When the concentration was raised from 100 to 150 g L -1 , the sprouting was substantially decreased between 1-100% in the stem and 48-100% in the flower extract among the indicator plants, while it was 61-100% in the leaf extract (Figs 1 & 2). Among them, leaf extract was more affected in many crops than stem and flower extract. On the other hand, germination (%), radicle, and hypocotyl length

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were significantly decreased at 50 to 100 g L -1 leaf extracts (Table 1). Increasing the concentration level eventually reduced the germination percentage over time. Both extracts of Parthenium inhibit the germination percentage of examined indicator for both weed species (Fig 2).
Methanol extracts had a significant allelopathic potential that influenced all the crops on radicle and hypocotyl length studied at varied doses except sweet gourd, cucumber, and maize. Extraction of the leaf at doses more than or equal to 50 g L -1 substantially decreased the radicle length of target plants (p�0.05) ( Table 1). With 100 to 150 g L -1 stem and flower extract, root development of certain plants was decreased by more than half, whereas the uppermost concentration of the leaf extract (100 to 150 g L -1 ) resulted in no root development such as Bambara groundnut, radish, pepper, okra, etc (Table 1). From the concentration level of 100 to 150 g L -1 Parthenium extract radicle length showed the inhibition level of 53-100, 36-100, and 10-100% from leaf, stem, and flower, respectively (Tables 2-4). As a result, the leaf extract had a higher concentration than the others (Table 2).
Furthermore, we observed that the weed crabgrass and goosegrass were severely affected by leaf and flower methanol extract in the doses of 50 to 150 g L -1 , but stem extract was affected by doses of 100 to 150 g L -1 . So, it was observed that severely affected weed by leaf than flower and stem plant parts. The amount of inhibition rose when the concentration level was raised. Different components of Parthenium reduced the shoot length of all examined plants by 27-100, 61-100 and 38-100%, respectively, at the doses of 100 to 150 g L -1 . Table 5 showed the half inhibitory (EC 50 ) impact of Parthenium plant parts with methanol extracts, as well as the sensitivity of the evaluated starting growth parameters and plants. The efficacy of stem extract (1554) was lower than that of leaf extract (811), and it was followed by flower extract (1109) in all tested crops. The EC 50 value showed some differences in sensitivity between the tested plant's responses to the inhibitory influence of P. hysterophorus (Table 5). In the case of leaf methanol extract maize, cucumber and sweet gourd was only impacted at higher concentrations. The rank value of these crops is 463, 144, and 108 respectively, which means that these crops are more tolerant, which shows that more doses need to destroy these plants. On the other hand, Bambara groundnut, radish, tomato, carrot, crabgrass, and goosegrass are more sensitive to leaf methanol extract next to pepper (53) and okra (41).

The half inhibitory effect of Parthenium methanol extracts
Again, in the case of stem methanol extract cucumber (277), maize (244), radish (227), okra (211), sweet gourd (165) and pepper (112) are more tolerant and other crops are more sensitive. It was inhibited by the extract. On the contrary, in the case of flower extract cucumber (264), maize (258), Bambara groundnut (200), and sweet gourd (151) are more tolerant than tomato, crabgrass, and goosegrass with other crops are more sensitive. These findings revealed that P. hysterophorys leaf extract had a greater effect on plant development than flower and stem extract at all dosages. Again, germination was seriously affected (163, 449, and 282) among the leaf, stem, and flower extracts indices, while radicle length (199, 667, and 449) and hypocotyl length (448, 437, and 377) were less affected to both plant sections. Overall, the methanol leaf extract of P. hysterophorus was very hazardous to all plants examined, particularly to germination, which was hindered at the lowest dosage.

Cluster and Principal Component Analysis (PCA)
Cluster analysis was also used to categorize distinct groups of plants with comparable responses to the inhibition of leaf, stem, and flower extracts by combining all three characteristics examined. Cluster analysis produced a dendrogram that revealed variation in sensitivity among the plants (Fig 3). Plants may be divided into four classes based on how they react to leaf, stem, and flower extracts ( Table 6). According to Table 6, group IV comprises tolerant monocot plants, whereas the dicot plants examined are referred to as the sensitive groups. Maize was recorded tolerant, whereas the moderately sweet gourd, cucumber, and carrot had an intermediate reaction to the allelopathic potential. The most vulnerable plants, on the other side, were Bambara groundnut, radish, tomato, crabgrass, goosegrass, okra, and pepper. Overall, the dicot plants were shown to be more active against the Parthenium extract than the monocots.
The principal component analysis (PCA), on the other hand, is a re-validation tool for cluster analysis. Johnson uses PCA to estimate the total variation that exists in a set of characters. As shown by the eigenvector in the two-dimensional (Fig 4) and three-dimensional (Fig 5) graphical elucidations, the majority of the indicator plants were spread at short distances, while just two were dispersed at long distances. Bambara groundnut and Maize were the accessions that were farthest from the centroid, whilst other accessions were close to it.

Identified phenolic derivatives from LC-MS analysis
The identified phenolic derivatives of P. hysterophorus plant parts with methanolic extracts through LC-MS analysis are listed in Table 1. The leaf, stem, and flower extracts of P. hysterophorus have diverse chemical compositions. A total of 7 Phenolic derivatives were detected from methanol extract of P. hysterophorus in different parts through LC-MS analysis (Table 1; Fig 6). These phenolic derivatives are responsible for inhibition of other plants, autotoxic, and

Inhibition of germination (%) Length of the radicle (cm) Length of the hypocotyl (cm)
Bambara dermatitis. Parthenin and other phenolic acids found in the leaf and flower extracts include vanillic acid, caffeic acid, quinic acid, anisic acid, chlorogenic acid, and ferulic acid, contrary Parthenin, vanillic acid found in the stem extract. The amount and kind of chemicals discovered in each plant were found to be proportional to herbicidal action. As a consequence, the compound of the various plant parts inhibited indicator plant germination and seedling growth, with the extraction of the leaf having a greater inhibitory influence than the other plant parts.  [19] reported that the extraction of different active phytochemicals with flavonoid concentration works best-using methanol as an extraction solvent. The germination of V. radiata seeds was tested for up to 120 hours using methanol crude The mean and standard error are used to express the data. The means for each extract with the same letters in the column are not substantially different at p�0.05.

Discussion
Inhibition percentages relative to the control are shown inside the parenthesis.
https://doi.org/10.1371/journal.pone.0280159.t003 Parthenium extracts and it was discovered that there is a considerable difference in germination kinetics between the treatments of methanol crudes [38]. Tef germination was significantly reduced at intermediate to higher concentrations when Parthenium flower and leaf extracts were used. This suggests that inhibitory compounds are present in larger concentrations in flower and leaf than in stem and root sections [39,40]. The fact that roots came into direct touch with the extract and then with inhibitory compounds, as reported in previous research with a variety of crops and weeds [41,42].

Length of the hypocotyl (cm) Inhibition of germination (%) Length of the radicle (cm) Length of the hypocotyl (cm)
The aerial parts extract of P. hysterophorus had a substantial influence on the germination of seed, radicle and hypocotyl length reduction in this investigation. These effects grew stronger as the concentration level increased. These discoveries are consistent with those of Wakjira et al. [43] and Mersie and Singh [44] who discovered a robust link between greater P. hysterophorus aqueous extract concentrations and increased poisonousness to agronomic crops and weeds. The effects of secondary metabolites generated by P. hysterophorus aerial parts on growth and development in Bambara groundnut weeds and chosen species. Phytochemicals isolated from P. hysterophorus stems, leaves, and flowers methanol extracts were competent to alter crops and weed seedling sprouting and development. Similarly, Motmainna et al. [45] discovered that P. hysterophorus extract had a considerable impact on the germination and development of the weed species. The degree of inhibition was raised when the concentration

Crops
Dose (g L -1 ) Flower extract The mean and standard error are used to express the data. The means for each extract with the same letters in the column are not substantially different at p�0.05.

Length of the hypocotyl (cm) Inhibition of germination (%) Length of the radicle (cm) Length of the hypocotyl (cm)
Inhibition percentages relative to the control are shown inside the parenthesis. https://doi.org/10.1371/journal.pone.0280159.t004

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of the extract was increased. Radicle growth is more vulnerable to allelopathic plant extracts than other organs due to radicles being the first tissue to be shown to be phytotoxic chemicals and have a more absorbent tissue than other parts [46,47], and/or the root apical meristem has a low mitotic division rate [48]. Furthermore, allelopathic elements can suppress the production of radicle and epidermis by altering genes involved in cellular characterization [49]. Parthenium extract was more effective than the B. alata and C. rutidosperma extract [45]. This is inconsistent with [50], who discovered that extracts of the allelopathic plant have a stronger effect on radicle length than hypocotyl development. This could be due to the roots being the initial ones to attract allelochemical substances from the atmosphere. The survivability rate of the target plants was inhibited by varying doses of P. hysterophorus leaf, stem, and flower methanol extracts. Maximum doses of methanol extracts included more inhibitory chemicals, resulting in more inhibition. In the same way, Han et al. [51] reported that the allelopathic potential of P. hysterophorus extracts was concentration-dependent, and allelopathic potential rose as extract concentration was raised. It was also claimed that the leaf extract had a more inhibitory allelopathic activity than other vegetative portions, and phytochemical research had already revealed a larger accumulation of growth inhibitors in P. hysterophorus leaves [52]. At all doses examined, the extracts inhibited P. minor germination, and when extract concentrations increased then inhibition increased [35]. However, extracts from the leaves had a higher level of toxicity than extracts from the stem [53].
Different plant species' susceptibility to inhibitory chemicals has been documented for a variety of causes. Msafiri et al. [54] observed that both tested species showed substantial allelopathic effects of P. hysterophorus seed and leaf aqueous extract on seed sprouting, root and hypocotyl length, and fresh and dry mass. According to Kobayasi [55], each species' have biological characteristics. The seed structure and seed coat penetrability can also play a role in  different reactions to similar allelopathic extracts [56]. Higher concentration reduced the seedling length of all the test crops but, sweet gourd, maize, and cucumber were less sensitive than other crops. This may be due to genotypic variation in response to the higher concentration of extracts. Similar results of the inhibitory effect were observed by Aslani et al. [57]. These findings are confirmed by the authors' other findings who stated that phytotoxic compounds are more vulnerable to smaller plants, although phytotoxin reactions differed from species to species [58].

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The phytochemical screening revealed a huge number of compounds in the P. hysterophorus extracts, some of which have previously been identified as poisons in several investigations [59,60]. Furthermore, various plant sections of P. hysterophorus contained a different number of compounds. The quantity of toxic compounds was more in the leaf than in the other plant parts; as a result, the leaves have a stronger inhibitory effect. P. hysterophorus leaves release allelochemicals into the soil by leaching or decomposition, and have the potential to impair the development of other plants by altering the physicochemical properties of soil, according to Dogra & Sood [61]. Arowosegbe & Afolayan [62] also found that beetroot (Beta vulgaris L.), Turnip (Brassica rapa L.), and carrot (D. carota L.) were all inhibited more by Aloe ferox Mill. leaf than by the root extract. The suppressive influence of extracts, according to Verdeguer et al. [63] is determined by the extract's chemical makeup as well as the plant sections to which it is applied. These findings are consistent with those of Javaid and Anjum [64] and Verma et al. [65] who discovered that parthenin and other phenolic acids such as caffeic acid, vanillic acid, anisic acid, chlorogenic acid, and para hydroxybenzoic acid are the most responsible for plant growth inhibition.

Conclusions
The results and discussion of the study revealed that herbicides made from natural molecules are cost-effective and environmentally friendly alternatives to synthetic chemical herbicides for controlling weeds in the crop field. To fulfil the objectives of the study, six concentrations (i.e., 25, 50, 75, 100, and 150 g L -1 ) of methanol extracts were isolated from P. hysterophorus leaf, stem and flower and were compared to the control (distilled water). The concentration of methanol extracts (isolated from P. hysterophorus leaf, stem, and flower); while increased, the rate of seed germination and seedling growth of Vigna subterranea (L.) Verdc, Raphanus sativus (L.) Domin, Cucurbita maxima Duchesne., Cucumis sativus L., Solanum lycopersicum L., Capsicum frutescens L., Zea mays L., Abelmoschus esculentus (L.) Moench, Daucus carota L., Digitaria sanguinalis (L.) Scop and Eleusine indica (L.) Gaertn decreased drastically, indicating that these methanol extracts have allelopathic potential. The allelopathic potentiality leaf extract of P. hysterophorus was found higher than the extraction of the stem (1554) and flower (1109), which is confirmed by EC 50 values, the principal component analysis (PCA) and LC-MS analysis, it is due to the leaf extract of P. hysterophorus contained 7 phenolic compounds which were responsible for inhibition of tested crops and weeds. Therefore, it can be concluded that the leaf phenolic compounds of P. hysterophorus may be used as a valuable natural herbicide in the future for the sustainability of crop production by controlling weeds.